Fig. 2

25HC interacts with α5β1 and αvβ3 integrins. a, b Biotinylation reactions were performed in the presence of either 25HC or DMSO (control). Cell lysates collected from BMDMs (a) or RAW 264.7 macrophages (b) incubated with biotinylated 25HC (biotin-25HC) or DMSO at 4 °C were precipitated (PPT) with avidin–agarose and subsequently immuno-blotted (IB) with either α5 integrin (a) or αv integrin (b) antibody. Total lysates were also blotted with α5 integrin (a), αv integrin (b), and actin (a, b) antibodies. c, d Biotin-25HC and DMSO (control) were incubated (PPT; precipitated) with avidin–agarose beads. 25HC conjugated beads were then incubated either with purified α5β1 integrin (c) or αvβ3 integrin (d). The avidin–agarose bound complex was subsequently immuno-blotted with either α5 integrin (c) or αv integrin (d) antibody. e, f Protein G-agarose beads were conjugated with either control IgG or antibody (Ab) specific for α5 integrin (e) and αv integrin (f). The beads were then incubated with either purified α5β1 integrin (e) or αvβ3 integrin (f) protein. Control beads (control IgG + purified integrin protein) and integrin protein bound beads (integrin-specific antibody + purified integrin protein) was incubated with tritiated ³H-25HC. Bead-bound radioactivity (count per minute; CPM) was measured by scintillation counter. g Cell lysate collected from RAW 264.7 macrophages incubated with biotin-25HC, biotinylated 27-hydroxycholesterol (biotin-27HC), biotinylated 4β-hydroxycholesterol (biotin-4βHC) or DMSO (control) at 4 °C was precipitated (PPT) with avidin–agarose and subsequently immuno-blotted with α5 integrin antibody. Total cell lysate was also blotted with α5 integrin and β-actin antibodies. h Surface plasmon resonance (SPR) analyses were performed by injecting increasing concentrations of 25HC (16 nM, 40 nM, 160 nM, 640 nM, 1.6 µM) into both the αvβ3 integrin-immobilized and control surface of the CM5 chips. Each background-corrected sensorgram is a representative of replicate runs. i Binding of radioactive ³H-25HC to immobilized αvβ3 integrin protein in the presence or absence of purified chemokine domain of fractalkine protein was assessed as described in the “methods” section. Immunoblot images are representative from three independent experiments. The radioactive values (mean ± standard deviation) are representative from two independent experiments. *p ≤ 0.05 using a Student’s t-test